Infection with the hepatitis C virus is a worrying health problem that has long been recognized, in particular in blood transfusion.
To reduce post-transfusion risks, it is necessary to detect the presence of the virus itself, before antibodies appear, and as soon as possible after contamination. This period between contamination and seroconversion (i.e., the appearance of antibodies) is called the “serological window”.
With a view to having a method that is simple, sensitive, specific, reproducible, inexpensive and easy to use, and can be automated for mass screening for detecting, firstly, the HCV antigen during the serological window period, and then for monitoring the serological evolution of the patient after seroconversion, detection of the anti-HCV antibodies, combined with detection of an HCV antigen, has been proposed.
However, this poses a major problem, that of interference between the anti-HCV antibodies present in the serum and labelled anti-HCV antibodies. Thus, the introduction on a solid phase, for the purpose of detecting a given antibody, of a target antigen having the same epitopes as those recognized by the labelled antibody or antibodies used for simultaneous, sandwich detection of an antigen would lead irreversibly to fixation of labelled antibody/antibodies on the solid phase and therefore a false positive response of the test.
This is particularly true in a system for simultaneous detection, on the same solid phase, of anti-HCV capsid antibodies and of HCV capsid antigen. Thus, deposition on the solid phase, for the purpose of detecting anti-HCV capsid antibodies, of a capsid antigen that has the same epitopes as those recognized by the labelled anti-HCV capsid antibody or antibodies used for detecting the capsid antigen leads to fixation of labelled antibody/antibodies on the solid phase and results in a false positive response of the test.
To tackle the problem of interference, application EP 1 020 727 (Advanced Life Science Institute) proposes a method for simultaneous measurement of the HCV capsid antigen and of anti-HCV capsid antibodies (a test of the “Combo” type), in which the antigen is captured and labelled by antibodies directed against capsid epitopes different from the capsid epitopes serving simultaneously for capture and detection of anti-capsid antibodies. A representative example is given where, in the simultaneous assay for sandwich detection of the antigen and of the antibodies in indirect assay, for detecting the antigen, a first (capture) antibody is used, directed against the epitopes of the sequence from amino acid (AA) 100 to amino acid 130 of the HCV capsid, and a second (detection) antibody, directed against the epitopes of the sequence AA40-50, and, for detecting the antibodies, the capture antigen used contains, for its part, the sequences AA1-42 and AA66-80.
Patent application WO 01/96875 (CHIRON) describes, among others, an assay for simultaneous detection of the capsid and of the anti-NS3 and NS4 antibodies, employing N-laurylsarcosine as detergent. It mentions an assay for simultaneous detection of the capsid antigen (in sandwich) and of anti-capsid and anti-non-structural HCV protein antibodies (in sandwich, double-antigen). For capturing the antigen, two antibodies are used, c11-3 and c11-7, which are reputed to recognize an extensive N-terminal portion (AA 10-53) of the HCV capsid, and for detection, a third antibody, c11-14, which is reputed to recognize a C-terminal portion (AA 120-130) of the HCV capsid. For detecting the antibodies, the capture antigen used is a fusion antigen with multiple epitopes which contains, fused with a fragment of superoxide dismutase (“SOD”), antigens NS3, NS4, NS5 and series of the capsid sequences from several strains of HCV: AA 9-53, bearing the R47L mutation, AA 64-88 and AA 67-84.
Patent application EP 1 251 353 (Ortho-Clinical Diagnostics) describes a “Combo complete” assay using the same antibodies for detecting the capsid, but without stating their origin or their epitope specificity. The anti-capsid antibodies are detected by means of a capsid antigen that has been modified (by mutagenesis): C22KSNV47, 48 (fusion protein with SOD comprising the deleted capsid sequence AA 10-99 of amino acids 47 and 48) or C22KSR47L (fusion protein with SOD comprising the capsid sequence AA 10-99, with a leucine replacing an arginine in position 47). Patent application WO2003/002749 (Abbott) describes many antigens and assays for detecting the HCV capsid antigen. The only “Combo complete” assay that it describes, under the name of “Real Combo”, employs a biotinylated peptide corresponding to amino acids 11-28 of the capsid, immobilized in the solid phase, for detecting anti-capsid antibodies. For detecting the capsid, it employs the combination of Advanced Life Science Institute antibody C11-14 (recognizing the capsid sequence AA 45-50) in the solid phase and C11-10 (recognizing the capsid sequence AA 32-36) labelled with acridine. Application WO 03/002749 therefore performs capture of the capsid antigen and capture of the anti-capsid antibodies via two capsid sites that are clearly separate, i.e., not superposed (AA 11-28 for detecting the antibodies and anti-AA 32-36 antibodies and AA 45-50 for detecting the antigen).
Patent application EP 1 310 512 (Ortho-Clinical Diagnostics) describes the use of peptides containing a mutated capsid sequence, eliminating the possibility of binding to HCV-specific mouse monoclonal antibodies, used in a test for detecting HCV infection.
The authors of the invention described in patent application WO2003/095968 made certain epitopes of the target antigens used for capturing the antibodies artificially different, by structural modification. The epitopes thus modified are then destroyed. Simultaneously, the antibodies used for capturing and/or detecting the antigens are for their part selected so that they recognize precisely unmodified epitopes present on the patient's antigens, and so that they therefore cannot bind to the modified antigens, which no longer have these same epitopes. Since the epitopes are no longer identical, there is no longer competition between the antibodies used for capturing and/or detecting the HCV antigen and the patient's antibodies. Patent application WO2003/095968 more precisely describes an HCV capsid peptide mutated in at least two separate epitope sites, and notably the peptide 1-75 (G34-G44-G47).